The present invention provides a process for the preparation of K-252a and intermediates useful in the process.
K-252a (methyl (15S,16R,18R)-16-hydroxy-15-methyl-3-oxo-28-oxa4,14,19-triazaoctacyclo[12.11.2.115,18.02,6.07,27.08,13.019,26.020,25]octacosa-1,6,8,10,12,20,22,24,26-nonaene-16-carboxylate) is a physiologically active substance produced by microorganisms which has demonstrated various pharmacological properties such as the ability to inhibit protein kinase C activity. Previously disclosed methods of isolating K-252a from microorganisms have proven to be inefficient when carried out on large amounts of material and have also been shown to generate the final product in low purity.
Alternatively, crude K-252a can be hydrolyzed to provide K-252b (which is more easily purified) and subsequently alkylated to provide K-252a of high purity. This method has also shown to be impractical for the production of large amounts of material, however, due to the required column chromatography and the use of dimethylsulfate as an alkylating agent, which is highly toxic.
As shown by these examples, there is a continuing need for methods of preparing K-252a of high purity that are amenable to large-scale production. The present invention discloses a preparation of K-252a which eliminates both the need for column chromatography and the use of dimethylsulfate.
In its principle embodiment, the present invention provides a process for preparing the compound of formula (II) 
wherein X is selected from the group consisting of Na, NH4, Li, and K; the process comprising:
(a) treating the compound of formula (I) 
with an aqueous hydroxide base, preferably an aqueous hydroxide base containing up to 25% organic solvent, more preferably an aqueous hydroxide base containing 20% acetone; most preferably aqueous sodium hydroxide or aqueous ammonium hydroxide containing 20% acetone;
(b) crystallizing the product of step (a); and
(c) isolating the product of step (b).
In another embodiment, the present invention provides a process for preparing the compound of formula (II), the process comprising:
(a) treating the compound of formula (I) with aqueous sodium hydroxide;
(b) cooling the product of step (a);
(c) optionally partially concentrating the product of step (b); and
(d) isolating the product of step (b) or step (c) by filtration.
In another embodiment, the present invention provides a process for preparing the compound of formula (III) 
the process comprising:
(a) dissolving the compound of formula (II) 
in a solvent, preferably N,N-dimethylacetamide or N,N-dimethylformamide, most preferably N,N-dimethylacetamide;
(b) optionally treating the solution of step (a) with a base, preferably sodium bicarbonate, sodium carbonate, or potassium carbonate, most preferably sodium bicarbonate;
(c) treating the solution of step (a) or step (b) with methyl p-toluenesulfonate; and
(d) isolating the product of step (c).
In another embodiment the present invention provides a process for preparing the compound of formula (III) 
the process comprising:
(a) treating the compound of formula (I) 
with an aqueous hydroxide base, preferably an aqueous hydroxide base containing up to 25% organic solvent, more preferably an aqueous hydroxide base containing 20% acetone; most preferably aqueous sodium hydroxide or aqueous ammonium hydroxide containing 20% acetone;
(b) crystallizing the product of step (a);
(c) isolating the product of step (b);
(d) dissolving the product of step (c) in a solvent, preferably N,N-dimethylacetamide or N,N-dimethylformamide, most preferably N,N-dimethylacetamide;
(e) optionally treating the solution of step (d) with a base, preferably sodium carbonate, sodium bicarbonate, or potassium carbonate, most preferably sodium bicarbonate; and
(f) reacting the product of step (d) or step (e) with a methylating agent, preferably methyl p-toluenesulfonate.
The present invention is directed to the preparation of K-252a (methyl (15S,16R,18R)-16-hydroxy-15-methyl-3-oxo-28-oxa-4,14,19-triazaoctacyclo[12.11.2.115,18.02,6.07.27.08,13.019,26.020,25]octacosa-1,6,8,10,12,20,22,24,26-nonaene-16-carboxylate) and to intermediates which are useful in this process. As used in the present specification the following terms have the meanings specified:
The term xe2x80x9caqueous,xe2x80x9d as used herein, refers to a solution that contains up to 30% organic solvent wherein the remaining solution is water. Preferred aqueous solutions contain up to 25% organic solvent, and more preferred aqueous solutions contain up to 20% organic solvent. Most preferred aqueous solutions contain 20% acetone.
The term xe2x80x9cbase,xe2x80x9d as used herein, refers to a reagent capable of accepting protons during the course of a reaction. Examples of bases include carbonate salts such as potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, and cesium carbonate; halides such as cesium fluoride; phosphates such as potassium phosphate, potassium dihydrogen phosphate, and potassium hydrogen phosphate; hydroxides such as lithium hydroxide, sodium hydroxide, ammonium hydroxide, and potassium hydroxide; disilylamides such as lithium hexamethyldisilazide, potassium hexamethyldisilazide, and sodium hexamethyldisilazide; trialkylamines such as triethylamine, diisopropylamine, and diisopropylethylamine; heterocyclic amines such as imidazole, pyridine, pyridazine, pyrimidine, and pyrazine; bicyclic amines such as DBN (1,5-diazabicyclo[4.3.0]non-5-ene) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene); and hydrides such as lithium hydride, sodium hydride, and potassium hydride. The base chosen for a particular conversion depends on the nature of the starting materials, the solvent or solvents in which the reaction is conducted, and the temperature at which the reaction is conducted.
The term xe2x80x9chydroxide base,xe2x80x9d as used herein, refers to lithium hydroxide, sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
The term xe2x80x9csolvent,xe2x80x9d as used herein, refers to an organic substance that is a liquid at between about 20 and about 35xc2x0 C. and does not interact with starting materials, reagents, intermediates, or products in a manner which adversely affects the yield of the desired product.
All of the processes of the present invention can be conducted as continuous processes. The term xe2x80x9ccontinuous process,xe2x80x9d as used herein, represents steps conducted without isolation of the intermediates.

Scheme 1 shows the synthesis of the compound of formula (III) (K-252a). The compound of formula (I) (K-252b; prepared according to a modification of the procedures described in J. Antibiotics 1986, 39, 1059-1065 and Biosci. Biotechnol. Biochem. 1998, 62, 1627-1629) can be treated with an aqueous hydroxide base to provide the compound of formula (II). Preferably, the aqueous hydroxide is sodium hydroxide or ammonium hydroxide. Examples of solvents used in this reaction include water, acetone, and mixtures thereof. The reaction is typically conducted at temperatures between about 20xc2x0 C. and about 55xc2x0 C. (depending on the solvent system used) and then cooled to a temperature of between about xe2x88x925xc2x0 C. and about 5xc2x0 C. to induce crystallization. The resulting crystals are then isolated by filtration to provide the compound of formula (II). Reaction times are typically about 10 to about 24 hours. In a preferred embodiment, the compound of formula (I) is treated with aqueous sodium hydroxide, stirred for one hour at room temperature, treated with acetone, cooled to about 0xc2x0 C. for about 19 hours, partially concentrated, and filtered to provide the compound of formula (II).
The compound of formula (II) can be converted to the compound of formula (III) (K-252a) by treatment with a methylating agent, preferably methyl p-toluenesulfonate, optionally in the presence of a base (to deprotonate any undesired compound of formula (I) remaining in the reaction). Representative bases used in this reaction include sodium bicarbonate, sodium carbonate, and potassium carbonate. When methyl p-toluenesulfonate is used, sodium acetate is added to decompose any remaining reagent. Examples of solvents used in this reaction include N,N-dimethylacetamide and N,N-dimethylformamide. The preferred solvent is N,N-dimethylacetamide. The reaction is typically conducted at about 35xc2x0 C. to about 45xc2x0 C. and then cooled to a temperature of between about 5xc2x0 C. and about 30xc2x0 C. Water is added to precipitate the product which is then isolated by filtration to provide the compound of formula (III). Reaction times are typically about 6 to about 12 hours.